The ADP-ribosylation factors (ARFs) are a family of GTP binding proteins that regulate membrane traffic and organelle structure in the cell. We have been studying the cellular function of ARF6, an ARF that affects plasma membrane (PM) traffic and the actin cytoskeleton. ARF6 regulates the movement of PM into and out of a novel, endosomal recycling pathway. The return of membrane to the PM requires ARF6 activation, occurs at discrete sites along the peripheral edges of cells and is associated with the formation of actin containing protrusions and membrane ruffling. Formation of protrusions is exaggerated in HeLa cells overexpressing ARF6 upon the addition of aluminum fluoride (AlF), an activator of heterotrimeric G proteins. The AlF treatment results in an accumulation of ARF6 at the PM, formation of protrusive structures, and a block in internalization of PM into the endosomal compartment. These are also characteristics of the effect of expressing the constitutively active mutant of ARF6 in cells. To investigate whether a G protein was responsible for this effect of AlF and thus a potential upstream regulator of ARF6, we cotransfected HeLa cells with ARF6 and constitutively active, candidate G alpha proteins. G alpha q, but not Gs, Gi2, or G12, appears to specifically activate ARF6 and recreates effects observed with AlF. We are currently investigating the mechanism whereby Gq maintains ARF6 in the activated state. In order to identify domains in ARF6 responsible for its cellular localization and function, we have constructed chimeras between ARF6 and ARF1 and expressed them in HeLa cells. ARF1 associates with Golgi membranes and regulates traffic through and maintenance of the Golgi complex. We found that the carboxyl- terminal halves of ARF6 and ARF1 contain information for targeting them to the correct membrane compartment, whereas the amino-terminal halves contain information for coupling to effector functions. Although the 1-6 chimera (amino-terminal half of ARF1, carboxyl-terminal half of ARF6) resembles ARF6 in it localization, it does not induce protrusions upon the addition of AlF. However, substitution of 2 amino acid residues (QS) in the amino-terminal half of this 1-6 chimera results in a gain-of- function, where protrusions now form. Mapping this effector domain to these two amino acids in ARF6 will facilitate the identification of interacting effector molecules and development of inhibitory peptides or antibodies that can be used to block ARF6 function.